Sustained release of antiinfectives

ABSTRACT

Provided, among other things, is a method of treating or ameliorating pulmonary infection in a cystic fibrosis patient comprising pulmonary administration of an effective amount of a liposomal/complexed antiinfective to the patient, wherein the (i) administrated amount is 50% or less of the comparative free drug amount, or (ii) the dosing is once a day or less, or (iii) both.

This application claims the priority of U.S. Provisional Application60/421,923, filed Oct. 29, 2002.

Certain sustained release technology suitable, for example, foradministration by inhalation employs liposomes and lipid complexes toprovide prolonged therapeutic effect of drug in the lung andsystemically by sustained release and the ability to target and enhancethe uptake of drug into sites of disease. The present inventioncomprises a liposomal antiinfective, and methods for treatment ofpulmonary infections in cystic fibrosis (CF) patients using liposomal orlipid-complexed antiinfective. Unexpectedly, treatments with the newformulation require a significantly lower dosage than that known to haveefficacy in the art.

As reported in Goodman and Gilman's The Pharmaceutical Basis ofTherapeutics, Eighth Edition, “Since the incidence of nephrotoxicity andototoxicity is related to the concentration to which an aminoglycosideaccumulates, it is critical to reduce the maintenance dosage of thesedrugs in patients with impaired renal function.” Since aminoglycosidescan produce vestibular or auditory dysfunction and nephrotoxicityregardless of a patient's impairments, it is important generally toreduce maintenance dosages. The present invention provides dramaticreductions in maintenance dosages.

CF patients have thick mucous and/or sputum secretions in the lungs,frequent consequential infections, and biofilms resulting from bacterialcolonizations. All these fluids and materials create barriers toeffectively targeting infections with antiinfectives. The presentinvention overcomes these barriers, and even allows reduced dosing (inamount or frequency), thereby reducing the drug load on patients.

For lung infections generally, the dosing schedule provided by theinvention provides a means of reducing drug load.

SUMMARY OF THE INVENTION

Provided, among other things, is a method of treating or amelioratingpulmonary infection in a cystic fibrosis patient comprising pulmonaryadministration of an effective amount of a liposomal/complexedantiinfective to the patient, wherein the (i) administrated amount is50% or less of the comparative free drug amount, or (ii) the dosing isonce a day or less, or (iii) both.

Also provided is a method of treating or ameliorating pulmonaryinfection in an animal comprising pulmonary administration of aneffective amount of a liposomal/complexed antiinfective to the patient,wherein the (i) administrated amount is 50% or less of the comparativefree drug amount, and (ii) the dosing is once every two days or less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Cross sectional diagram of the sputum/biofilm seen in patientswith cystic fibrosis.

FIG. 2: Graphical representation of the targeting and depot effect ofthe drug of the present invention.

FIGS. 3 and 4: Graphical representations of bacteriology of amikacin invarious forms.

FIG. 5: Graphical representation of sustained release forliposomal/complexed amikacin and tobramycin.

FIG. 6: Data on free or complexed ciprofloxacin.

FIG. 7: Graphical representation of drug residence in the lung givenvarious dosing schedules.

DETAILED DESCRIPTION OF THE INVENTION

The present application discloses a method of treating or amelioratingpulmonary infections, such as in cystic fibrosis patients, comprisingadministration of antiinfective (such as antibiotic) encapsulated inlipid-based particles.

Antiinfectives are agents that act against infections, such asbacterial, mycobacterial, fungal, viral or protozoal infections.

Antiinfectives covered by the invention include but are not limited toaminoglycosides (e.g., streptomycin, gentamicin, tobramycin, amikacin,netilmicin, kanamycin, and the like), tetracyclines (such aschlortetracycline, oxytetracycline, methacycline, doxycycline,minocycline and the like), sulfonamides (e.g., sulfanilamide,sulfadiazine, sulfamethaoxazole, sulfisoxazole, sulfacetamide, and thelike), para-aminobenzoic acid, diaminopyrimidines (such as trimethoprim,often used in conjunction with sulfamethoxazole, pyrazinamide, and thelike), quinolones (such as nalidixic acid, cinoxacin, ciprofloxacin andnorfloxacin and the like), penicillins (such as penicillin G, penicillinV, ampicillin, amoxicillin, bacampicillin, carbenicillin, carbenicillinindanyl, ticarcillin, azlocillin, mezlocillin, piperacillin, and thelike), penicillinase resistant penicillin (such as methicillin,oxacillin, cloxacillin, dicloxacillin, nafcillin and the like), firstgeneration cephalosporins (such as cefadroxil, cephalexin, cephradine,cephalothin, cephapirin, cefazolin, and the like), second generationcephalosporins (such as cefaclor, cefamandole, cefonicid, cefoxitin,cefotetan, cefuroxime, cefuroxime axetil; cefmetazole, cefprozil,loracarbef, ceforanide, and the like), third generation cephalosporins(such as cefepime, cefoperazone, cefotaxime, ceftizoxime, ceftriaxone,ceftazidime, cefixime, cefpodoxime, ceftibuten, and the like), otherbeta-lactams (such as imipenem, meropenem, aztreonam, clavulanic acid,sulbactam, tazobactam, and the like), beta-lactamase inhibitors (such asclavulanic acid), chlorampheiicol, macrolides (such as erythromycin,azithromycin, clarithromycin, and the like), lincomycin, clindamycin,spectinomycin, polymyxin B, polymixins (such as polymyxin A, B, C, D,E₁(colistin A), or E₂, colistin B or C, and the like) colistin,vancomycin, bacitracin, isoniazid, rifampin, ethambutol, ethionamide,aminosalicylic acid, cycloserine, capreomycin, sulfones (such asdapsone, sulfoxone sodium, and the like), clofazimine, thalidomide, orany other antibacterial agent that can be lipid encapsulated.Antiinfectives can include antifungal agents, including polyeneantifungals (such as amphotericin B, nystatin, natamycin, and the like),flucytosine, imidazoles (such as miconazole, clotrimazole, econazole,ketoconazole, and the like), triazoles (such as itraconazole,fluconazole, and the like), griseofulvin, terconazole, butoconazoleciclopirax, ciclopirox olamine, haloprogin, tolnaftate, naftifine,terbinafine, or any other antifungal that can be lipid encapsulated orcomplexed. Discussion and the examples are directed primarily towardamikacin but the scope of the application is not intended to be limitedto this antiinfective. Combinations of drugs can be used.

Particularly preferred antiinfectives include the aminoglycosides, thequinolones, the polyene antifungals and the polymyxins.

Among the pulmonary infections (such as in cystic fibrosis patients)that can be treated with the methods of the invention are pseudomonas(e.g., P. aeruginosa, P. paucimobilis, P. putida, P. fluorescens, and P.acidovorans), staphylococcal, Methicillin-resistant Staphylococcusaureus (MRSA), streptococcal (including by Streptococcus pneumoniae),Escherichia coli, Klebsiella, Enterobacter, Serratia, Haemophilus,Yersinia pestis, Burkholderia pseudomallei, B. cepacia, B. gladioli, B.multivorans, B. vietnamiensis, Mycobacterium tuberculosis, M. aviumcomplex (MAC) (M. avium and M. intracellulare), M. kansasii, M. xenopi,M. marinum, M. ulcerans, or M. fortuitum complex (M. fortuitum and M.chelonei) infections.

In one preferred embodiment the present invention comprises a method oftreatment comprising administration of liposomal/complexed amikacin.

The “liposomal or lipid-complexed” antiinfective, or“liposomal/complexed” antiinfective, or “Lip-antiinfective,” or “Lip-An”discussed herein is any form of antiinfective composition where at leastabout 1% by weight of the antiinfective is associated with the lipideither as part of a complex with the lipid, or as a liposome where theantibiotic may be in the aqueous phase or the hydrophobic bilayer phaseor at the interfacial headgroup region of the liposomal bilayer.Preferably, at least about 5%, or at least about 10%, or at least about20%, or at least about 25%, is so associated. Association is measured byseparation through a filter where lipid and lipid-associated drug isretained and free drug is in the filtrate.

Treatment with liposomal/complexed antiinfective requires a notablylower dosage than prior known treatments. In one preferred embodimentless than 100 mg per day of an aminoglycoside is administered to humans.In another preferred embodiment approximately 30 to 50 mg isadministered every other day or every third day. It is expected thatdosages can be correspondingly lowered for other species as compared tothe dosage recommended for antiinfective that is not liposomal orlipid-complexed. This is an unexpectedly low dosage.

Where no specific dosage is provided below, the preferred dosage of theinvention is 50% or less, 35% or less, 20% or less, or 10% or less, ofthe minimum free drug (which of course can be a salt) amount that iseffective, if delivered to the lungs via a nebulizer, to reduce the CFUcount in the lungs by one order of magnitude over the course of a 14-daytreatment. The comparative free drug amount is the cumulative amountthat would be used in the dosing period applied with the drugadministration of the invention. The comparative minimum free drugdefined in this paragraph is a “comparative free drug amount.”

The non-CF treating embodiments of the invention can be used with anyanimal, though preferably with humans. Relative amounts in a givenanimal are measured with respect to such animal.

The dosing schedule is preferably once a day or less. In preferredembodiments, the dosing schedule is once every other day, every thirdday, every week, or less. For example, the dosing schedule can be everyother day or less, using 50% or less of the comparative free drugamount. Or, for example, the dosing can be daily using 35% or less ofthe comparative free drug amount.

To treat the infections of the invention, an effective amount of apharmaceutical compound will be recognized by clinicians but includes anamount effective to treat, reduce, ameliorate, eliminate or prevent oneor more symptoms of the disease sought to be treated or the conditionsought to be avoided or treated, or to otherwise produce a clinicallyrecognizable change in the pathology of the disease or condition.Amelioration includes reducing the incidence or severity of infectionsin animals treated prophylactically. In certain embodiments, theeffective amount is one effective to treat or ameliorate after symptomsof lung infection have arisen. In certain other embodiments, theeffective amount is one effective to treat or ameliorate the averageincidence or severity of infections in animals treated prophylactically(as measured by statistical studies).

Liposome or other lipid based delivery systems can be administered forinhalation either as a nebulized spray, powder, or aerosol, or byintrathecal administration. Inhalation administrations are preferred.The overall result is a less frequent administration and an enhancedtherapeutic index compared to free drug or parenteral form of the drug.Liposomes or lipid complexes are particularly advantageous due to theirability to protect the drug while being compatible with the lung liningor lung surfactant.

The present invention includes methods for treatment of pulmonarygram-negative infections. One usefully treated infection is chronicpseudomonal infection in CF patients. Known treatments of lunginfections (such as in CF patients) with amikacin generally compriseadministering approximately 200-600 mg of amikacin or tobramycin per dayvia inhalation. The present invention allows for treatment byadministering, in one preferred embodiment, 100 mg or less of amikacinper day (or normalized to 100 mg per day or less if dosing lessfrequent). In yet another embodiment administration of 60 mg or less ofamikacin every day is performed. And in still another embodimentadministration of approximately 30 to 50 mg not more than once every 2days is performed. The most preferred embodiment comprisesadministration of approximately 30 to 50 mg every other day or everythird day.

Known treatments of lung infections with tobramycin generally compriseadministering 300 mg, twice a day, in adults and children 6 years of ageor older. The present invention allows for treatment by administering,in one preferred embodiment, 100 mg or less of tobramycin per day. Inyet another embodiment administration of 60 mg or less of tobramycinevery day is performed. And in still another embodiment administrationof approximately 30 to 50 mg not more than once every 2 days isperformed. The most preferred embodiment comprises administration ofapproximately 30 to 50 mg every other day or every third day.

The lipids used in the compositions of the present invention can besynthetic, semi-synthetic or naturally-occurring lipids, includingphospholipids, tocopherols, steroids, fatty acids, glycoproteins such asalbumin, negatively-charged lipids and cationic lipids. Phosholipidsinclude egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG),egg phosphatidylinositol (EPI), egg phosphatidylserine (EPS),phosphatidylethanolamine (EPE), and egg phosphatidic acid (EPA); thesoya counterparts, soy phosphatidylcholine (SPC); SPG, SPS, SPI, SPE,and SPA; the hydrogenated egg and soya counterparts (e.g., HEPC, HSPC),other phospholipids made up of ester linkages of fatty acids in the 2and 3 of glycerol positions containing chains of 12 to 26 carbon atomsand different head groups in the 1 position of glycerol that includecholine, glycerol, inositol, serine, ethanolamine, as well as thecorresponding phosphatidic acids. The chains on these fatty acids can besaturated or unsaturated, and the phospholipid can be made up of fattyacids of different chain lengths and different degrees of unsaturation.In particular, the compositions of the formulations can includedipalmitoylphosphatidylcholine (DPPC), a major constituent ofnaturally-occurring lung surfactant as well asdioleoylphosphatidylcholine (DOPC). Other examples includedimyristoylphosphatidylcholine (DMPC) anddimyristoylphosphatidylglycerol (DMPG) dipalmitoylphosphatidcholine(DPPC) and dipalmitoylphosphatidylglycerol (DPPG)distearoylphosphatidylcholine (DSPC) and distearoylphosphatidylglycerol(DSPO), dioleylphosphatidylethanolamine (DOPE) and mixed phospholipidslike palmitoylstearoylphosphatidylcholine (PSPC) andpalmitoylstearoylphosphatidylglycerol (PSPG), triacylglycerol,diacylglycerol, seranide, sphingosine, sphingomyelin and single acylatedphospholipids like mono-oleoyl-phosphatidylethanolamine (MOPE).

The lipids used can include ammonium salts of fatty acids, phospholipidsand glycerides, steroids, phosphatidylglycerols (PGs), phosphatidicacids (PAs), phosphotidylcholines (PCs), phosphatidylinositols (PIs) andthe phosphatidylserines (PSs). The fatty acids include fatty acids ofcarbon chain lengths of 12 to 26 carbon atoms that are either saturatedor unsaturated. Some specific examples include: myristylamine,palmitylamine, laurylamine and stearylamine, dilauroylethylphosphocholine (DLEP), dimyristoyl ethylphosphocholine (DMEP),dipalmitoyl ethylphosphocholine (DPEP) and distearoylethylphosphocholine (DSEP), N-(2, 3-di-(9(Z)-octadecenyloxy)-prop-1-yl-N,N,N-trimethylammonium chloride (DOTMA)and 1, 2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP). Examples ofsteroids include cholesterol and ergosterol. Examples of PGs, PAs, PIs,PCs and PSs include DMPG, DPPG, DSPG, DMPA, DPPA, DSPA, DMPI, DPPI,DSPI, DMPS, DPPS and DSPS, DSPC, DPPC, DMPC, DOPC, egg PC.

Liposomes or lipid complexes composed of phosphatidylcholines, such asDPPC, aid in the uptake by the cells in the lung such as the alveolarmacrophages and helps to sustain release of the antiinfective agent inthe lung (Gonzales-Rothi et al. (1991)). The negatively charged lipidssuch as the PGs, PAs, PSs and PIs, in addition to reducing particleaggregation, can play a role in the sustained release characteristics ofthe inhalation formulation as well as in the transport of theformulation across the lung (transcytosis) for systemic uptake. Thesterol compounds are believed to affect the release and leakagecharacteristics of the formulation.

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes can be unilamellar vesicles(possessing a single membrane bilayer) or multilamellar vesicles(onion-like structures characterized by multiple membrane bilayers, eachseparated from the next by an aqueous layer). The bilayer is composed oftwo lipid monolayers having a hydrophobic “tail” region and ahydrophilic “head” region. The structure of the membrane bilayer is suchthat the hydrophobic (nonpolar) “tails” of the lipid monolayers orienttoward the center of the bilayer while the hydrophilic “heads” orienttowards the aqueous phase. Lipid complexes are associations betweenlipid and the antiinfective agent that is being incorporated. Thisassociation can be covalent, ionic, electrostatic, noncovalent, orsteric. These complexes are non-liposomal and are incapable ofentrapping additional water soluble solutes. Examples of such complexesinclude lipid complexes of amphotericin B (Janoff et al., Proc. NatAcad. Sci., 85:6122 6126, 1988) and cardiolipin complexed withdoxorubicin.

A lipid clathrate is a three-dimensional, cage-like structure employingone or more lipids wherein the structure entraps a bioactive agent. Suchclathrates are included in the scope of the present invention.

Proliposomes are formulations that can become liposomes or lipidcomplexes upon coming in contact with an aqueous liquid. Agitation orother mixing can be necessary. Such proliposomes are included in thescope of the present invention.

Liposomes can be produced by a variety of methods (for example, see,Bally, Cullis et al., Biotechnol Adv. 5(1):194, 1987). Bangham'sprocedure (J. Mol. Biol., J Mol Biol. 13(1):238-52, 1965) producesordinary multilamellar vesicles (MLVs). Lenk et al. (U.S. Pat. Nos.4,522,803, 5,030,453 and 5,169,637), Fountain et al. (U.S. Pat. No.4,588,578) and Cullis et al. (U.S. Pat. No. 4,975,282) disclose methodsfor producing multilamellar liposomes having substantially equalinterlamellar solute distribution in each of their aqueous compartments.Paphadjopoulos et al., U.S. Pat. No. 4,235,871, discloses preparation ofoligolamellar liposomes by reverse phase evaporation.

Unilamellar vesicles can be produced from MLVs by a number oftechniques, for example, the extrusion of Cullis et al. (U.S. Pat. No.5,008,050) and Loughrey et al. (U.S. Pat. No. 5,059,421). Sonication andhomogenization can be used to produce smaller unilamellar liposomes fromlarger liposomes (see, for example, Paphadjopoulos et al., Biochim.Biophys. Acta., 135:624-638, 1967; Deamer, U.S. Pat. No. 4,515,736; andChapman et al., Liposome Technol., 1984, pp. 1-18).

The original liposome preparation of Bangham et al. (J. Mol. Biol.,1965, 13:238-252) involves suspending phospholipids in an organicsolvent which is then evaporated to dryness leaving a phospholipid filmon the reaction vessel. Next, an appropriate amount of aqueous phase isadded, the mixture is allowed to “swell”, and the resulting liposomeswhich consist of multilamellar vesicles (MLVs) are dispersed bymechanical means. This preparation provides the basis for thedevelopment of the small sonicated unilamellar vesicles described byPapahadjopoulos et al. (Biochim. Biophys, Acta., 1967, 135:624-638), andlarge unilamellar vesicles.

Techniques for producing large unilamellar vesicles (LUVs), such as,reverse phase evaporation, infusion procedures, and detergent dilution,can be used to produce liposomes. A review of these and other methodsfor producing liposomes can be found in the text Liposomes, Marc Ostro,ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, the pertinentportions of which are incorporated herein by reference. See also Szoka,Jr. et al., (1980, Ann. Rev. Biophys. Bioeng., 9:467), the pertinentportions of which are also incorporated herein by reference.

Other techniques that are used to prepare vesicles include those thatform reverse-phase evaporation vesicles (REV), Papahadjopoulos et al.,U.S. Pat. No. 4,235,871. Another class of liposomes that can be used arethose characterized as having substantially equal lamellar solutedistribution. This class of liposomes is denominated as stableplurilamellar vesicles (SPLV) as defined in U.S. Pat. No. 4,522,803 toLenk, et al. and includes monophasic vesicles as described in U.S. Pat.No. 4,588,578 to Fountain, et al. and frozen and thawed multilamellarvesicles (FATMLV) as described above.

A variety of sterols and their water soluble derivatives such ascholesterol hemisuccinate have been used to form liposomes; seespecifically Janoff et al., U.S. Pat. No. 4,721,612, issued Jan. 26,1988, entitled “Steroidal Liposomes.” Mayhew et al, described a methodfor reducing the toxicity of antibacterial agents and antiviral agentsby encapsulating them in liposomes comprising alpha-tocopherol andcertain derivatives thereof. Also, a variety of tocopherols and theirwater soluble derivatives have been used to form liposomes, see Janoffet al., U.S. Pat. No. 5,041,278.

A process for forming liposomes or lipid complexes involves a “solventinfusion” process. This is a process that includes dissolving one ormore lipids in a small, preferably minimal, amount of a processcompatible solvent to form a lipid suspension or solution (preferably asolution) and then injecting the solution into an aqueous mediumcontaining bioactive agents. Typically a process compatible solvent isone that can be washed away in a aqueous process such as dialysis. Thecomposition that is cool/warm cycled is preferably formed by solventinfusion, with ethanol infusion being preferred. Alcohols are preferredas solvents. “Ethanol infusion,” a type of solvent infusion, is aprocess that includes dissolving one or more lipids in a small,preferably minimal, amount of ethanol to form a lipid solution and theninjecting the solution into an aqueous medium containing bioactiveagents. A “small” amount of solvent is an amount compatible with formingliposomes or lipid complexes in the infusion process. Such processes aredescribed in Lee et al., U.S. patent application Ser. No. 10/634,144,filed Aug. 4, 2003, Pilkiewicz et al, U.S. patent application Ser. No.10/383,173, filed Mar. 5, 2003, and Boni et al., U.S. patent applicationSer. No. 10/383,004, filed Mar. 5, 2003, which applications are herebyincorporated by reference in their entirety.

Liposome or lipid complex sizing can be accomplished by a number ofmethods, such as extrusion, sonication and homogenization techniqueswhich are well known, and readily practiced, by ordinarily skilledartisans. Extrusion involves passing liposomes, under pressure, one ormore times through filters having defined pore sizes. The filters aregenerally made of polycarbonate, but the filters may be made of anydurable material which does not interact with the liposomes and which issufficiently strong to allow extrusion under sufficient pressure.Preferred filters include “straight through” filters because theygenerally can withstand the higher pressure of the preferred extrusionprocesses of the present invention. “Tortuous path” filters may also beused. Extrusion can also use asymmetric filters, such as AnotecO™filters, which involves extruding liposomes through a branched-pore typealuminum oxide porous filter.

Liposomes or lipid complexes can also be size reduced by sonication,which employs sonic energy to disrupt or shear liposomes, which willspontaneously reform into smaller liposomes. Sonication is conducted byimmersing a glass tube containing the liposome suspension into the sonicepicenter produced in a bath-type sonicator. Alternatively, a probe typesonicator may be used in which the sonic energy is generated byvibration of a titanium probe in direct contact with the liposomesuspension. Homogenization and milling apparatii, such as the GiffordWood homogenizer, Polytron™ or Microfluidizer™, can also be used tobreak down larger liposomes or lipid complexes into smaller liposomes orlipid complexes.

The resulting liposomes/complexes can be separated into homogeneouspopulations using methods well known in the art; such as tangential flowfiltration. In this procedure, a heterogeneously sized population ofliposomes or lipid complexes is passed through tangential flow filters,thereby resulting in a liposome population with an upper and/or lowersize limit. When two filters of differing sizes, that is, havingdifferent pore diameters, are employed, liposomes smaller than the firstpore diameter pass through the filter. This filtrate can the be subjectto tangential flow filtration through a second filter, having a smallerpore size than the first filter. The retentate of this filter is aliposomal/complexe population having upper and lower size limits definedby the pore sizes of the first and second filters, respectively. Mayeret al. found that the problems associated with efficient entrapment oflipophilic ionizable bioactive agents such as antineoplastic agents, forexample, anthracyclines or vinca alkaloids, can be alleviated byemploying transmembrane ion gradients. Aside from inducing greateruptake, such transmembrane gradients can also act to increaseantiinfective retention in the liposomes/complexes.

Liposomal/complexed antiinfective has a sustained antiinfective effectand lower toxicity allowing less frequent administration and an enhancedtherapeutic index. In preclinical animal studies and in comparison toinhaled Tobramycin (not-liposomal or lipid-complexed) at the equivalentdose level, liposomal/complexed amikacin was shown to have, during thetime period shortly after administration to over 24 hours later, druglevels in the lung that ranged from two to several hundred times that ofTobramycin. Additionally, liposomal/complexed amikacin maintained theselevels for well over 24 hours. In an animal model designed to mimic thepseudomonas infection seen in CF patients, liposomal/complexed amikacinwas shown to significantly eliminate the infection in the animals' lungswhen compared to free aminoglycosides.

Lung surfactant allows for the expansion and compression of the lungsduring breathing. This is accomplished by coating the lung with acombination of lipid and protein. The lipid is presented as a monolayerwith the hydrophobic chains directed outward. The lipid represents 80%of the lung surfactant, the majority of the lipid beingphosphatidylcholine, 50% of which is dipalmitoyl phosphatidylcholine(DPPC) (Veldhuizen et al, 1998). The surfactant proteins (SP) that arepresent function to maintain structure and facilitate both expansion andcompression of the lung surfactant as occurs during breathing. Of these,SP-B and SP-C specifically have lytic behavior and can lyse liposomes(Hagwood et al., 1998; Johansson, 1998). This lytic behavior is believedto facilitate the gradual break-up of liposomes followed, by theirrelease of internal contents allowing for a depot effect. This break-upof liposomes occurs naturally as evidenced by the spontaneous unravelingof lamellar bodies ejected by exocytosis (Ikegami & Jobe, 1998) Inaddition to becoming assimilated within the lung surfactant, liposomescan be directly ingested by macrophages through phagocytosis (Couveur etal., 1991; Gonzales-Roth et al., 1991; Swenson et al, 1991). Uptake ofliposomes by alveolar macrophages is another means by which drugs can bedelivered to the diseased site.

The lipids preferably used to form either liposomes or lipid complexesfor inhalation use are common to the endogenous lipids found in the lungsurfactant. Liposomes are composed of bilayers that entrap the desiredpharmaceutical. These can be configured as multilamellar vesicles ofconcentric bilayers with the pharmaceutical trapped within either thelipid of the different layers or the aqueous space between the layers.The present invention unique processes to create unique liposomes andlipid/drug complexes. Both the processes and the product of theseprocesses are part of the present invention.

The lipid to drug ratio using the process of the present invention ispreferably less than 3 to 1. And more preferably the lipid to drug ratiois less than 2.5 to 1. Further the percentage of free antiinfective,present after the product is dialyzed for a particular duration, isdecreased.

All processes described herein can be easily adapted for large scale,aseptic manufacture. The final liposome size can be adjusted bymodifying the lipid composition, concentration, excipients, andprocessing parameters.

An obstacle to treating infectious diseases such as Pseudomonasaeruginosa, the leading cause of chronic illness in cystic fibrosispatients is drug penetration within the sputum/biofilm barrier onepithelial cells (FIG. 1). In FIG. 1, the donut shapes representliposomal/complexed antiinfective, the “+” symbol represents freeantiinfective, the “−” symbol mucin, alginate and DNA, and the solid barsymbol represents Pseudomonas aeruginosa. This barrier is composed ofboth colonized and planktonic P. aeruginosa embedded in alginate orexopolysaccharides from bacteria, as well as DNA from damagedleukocytes, and mucin from lung epithelial cells, all possessing a netnegative charge (Costerton, et al., 1999). This negative charge binds upand prevent penetration of positively charged drugs such asaminoglycosides, rendering them biologically ineffective (Mendelman etal., 1985). Entrapment of antiinfectives within liposomes or lipidcomplexes could shield or partially shield the antiinfectives fromnon-specific binding to the sputum/biofilm, allowing for liposomes orlipid complexes (with entrapped aminoglycoside) to penetrate (FIG. 1).

Amikacin has been shown to have a high degree of resistance to bacterialenzymes, thus providing a greater percent of susceptible clinicalisolates than found for other aminoglycosides including tobramycin andgentamicin (Price et al., 1976). In particular, P. aeruginosa isolatesare far more sensitive to amikacin than other aminoglycosides whileexhibiting no cross-resistance (Damaso et al., 1976).

The sustained release and depot effect of liposomal/complexed amikacinis clearly seen in FIG. 2. In this study rats were given tobramycin viaintratracheal and intravenous administration. The rats were also givenliposomal/complexed amikacin intratracheally at the same dose (4mg/rat). The data show that it is only with the liposomal/complexedamikacin that a sustained release and depot effect is achieved. In fact,24 hours after dosing, only liposomal/complexed amikacin showssignificant levels of the drug in the animal's lungs, while bothtobramycin formulations revealed negligible levels, primarily due, it isbelieved to rapid systemic absorption. This greater than a hundred-foldincrease of aminoglycoside in the lung for liposomal/complexedantiinfective supports the idea of a sustained releaseliposomal/complexed antiinfective that can be taken significantly lessoften than the currently approved TOBI™ formulation (Chiron Corporation,Ameryville, Calif.).

Moreover, the presence of a sputum/biofilm prevents the penetration ofthe free aminoglycosides due to binding of the antiinfectives to itssurface (FIG. 1). Therefore, doses in excess of 1000 μg oftobramycin/gram of lung tissue are needed to show a therapeutic effectin CF patients. This is overcome with liposomal/complexed amikacin.Thus, the therapeutic level of drug is maintained for a longer period oftime in the liposomal/complexed formulations of amikacin compared tofree tobramycin. This facilitation of binding and penetration could alsobe a means by which liposomal/complexed amikacin could significantlyreduce bacterial resistance commonly seen to develop when antibacterialsare present in vivo at levels below the minimum inhibitoryconcentration.

The pharmacokinetics of amikacin was determined in rats followingintratracheal (IT) administration of either free tobramycin orliposomal/complexed amikacin. These data were compared to thedistribution obtained in the lungs following a tail vein injection offree tobramycin. In all cases a dose of 4 mg/rat was administered. Ascan be seen in FIG. 2, a much larger deposition of aminoglycoside can bedelivered by IT compared to injection. The depot effect ofliposomal/complexed antiinfective technology is also demonstrated inthat in comparison to tobramycin given either IT or IV, a greater than ahundred-fold increase in drug for liposomal/complexed amikacin stillremains in the lungs twenty-four hours following administration. Thus,the therapeutic level of drug is maintained for a longer period of timein the liposomal formulations of amikacin compared to free tobramycin.

The binding of aminoglycosides to sputum of CF patients is a concern,particularly if this binding reduces the bioactivity of theantiinfective (Hunt et al., 1995). To determine whetherliposomal/complexed amikacin can retain biological activity over aprolonged period of time, normal rats were administeredliposomal/complexed amikacin by intratracheal instillation. This wasfollowed by its removal at 2 or 24 hours via a bronchial alveolar lavage(BAL) to determine biological activity. Samples were concentrated byultrafiltration followed by filtration (0.2 micron) to removecontaminating lung microbes. Amikacin concentration was determinedemploying a TDX instrument and biological activity determined using aMueller Hinton broth dilution assay (Pseudomonas aeruginosa). Theresults are shown in the following Table I:

time amakacin in BAL amakacin in filtrate MIC (hours) (microgram/mL)(microgram/mL) (μg/mL) 2 160 119 1.9 24 73 32 4.0

As shown by the above table, the recovered filtered liposomal/complexedamikacin was capable of killing P. aeruginosa in a Mueller Hinton brothassay even after 24 hours with an MIC of 4. At 2 hours an MIC of 2 wasobtained, which is similar to that obtained for the filteredliposomal/complexed amikacin stock. Thus, the liposomal/complexedamikacin was still active following 24 hours in the lung. At 24 hoursfree tobramycin at the same dose was undetectable in a BAL. Thisindicates that not only is the liposomal/complexed antiinfectiveformulation retained in the lung, but it is also freely available topenetrate a sputum/biofilm over time. These data combined with the factsas evident in FIG. 2 and Table II (below), that liposomal/complexedamikacin releases the free antiinfective over time while maintaininghigh levels of the antiinfective in the lungs, supports the rationalethat this system may yield a sustained antiinfective effect over time.This effect should prove significant in reducing both the bio-burden ofthe Pseudomonas and the development of resistance due to trough levelsof antiinfective.

As an in vitro demonstration of slow release of liposomal/complexedamikacin and its sustained antiinfective effect, the formulation wasincubated in sputum from patients with Chronic Obstructive PulmonaryDisease (COPD) containing PAO1 mucoid Pseudomonas. Theliposomal/complexed amikacin was also incubated in alginate containingPAO1 mucoid Pseudomonas. In both cases sustained and enhanced killing ofthe pseudomonas over time was observed, as shown in Table II:

In Vitro Sputum/Alginate Assay (% survival of PA01 Mucoid Pseudomonas)Incubation time at 37° C. Amikacin conc. 1 h 3 h 6 h 24 h (microgram/mL)Lip-An-15 Sputum 81 15 22 <1 8 Lip-An-15 Alginate 100 59 1 <1 10Classical kill curves are not applicable for liposomal/complexedantiinfective technology because the liposomal formulations exhibit aslow release of antiinfective with an enhanced antiinfective effect. Theliposome/complex protects the amikacin from the sputum and/or alginateuntil its release. In time, complete killing is observed, consistentwith slow release sustained antiinfective effect model with nointerference or inactivation of antiinfective.

The efficacy of liposomal/complexed amikacin formulations was studiedusing a model for chronic pulmonary infection (Cash et al., 1979) whereP. aeruginosa, embedded in an agarose bead matrix, was instilled in thetrachea of rats. This mucoid Pseudomonas animal model was developed toresemble the Pseudomonas infections seen in CF patients. Some of theclinical correlates to CF include: a similar lung pathology; thedevelopment of immune complex disorders; and a conversion to the mucoidphenotype by P. aeruginosa strains (Cantin and Woods, 1999). Rat lungswere infected with over 10⁷ CPUs of a mucoid Pseudomonas (strain PAO1)taken from a CF patient isolate, and subsequently treated with (a) freeaminoglycoside, (b) the lipid vehicle alone as non-drug control, and (c)liposomal/complexed amikacin. In addition, formulations were firstscreened on the ability to kill in vitro P. aeruginosa on modifiedKirby-Bauer plates.

Various liposomal/complexed amikacin formulations were tested based oneither different lipid compositions or manufacturing parametersresulting in different killing zones in in vitro experiments. Thisexperiment was designed to determine the increase in efficacy obtainedwith liposomal/complexed aminoglycoside over free aminoglycoside. Blankcontrol lipid compositions, two different liposomal/complexed amikacinformulations and free amikacin and free Tobramycin at the sameaminoglycoside concentrations as the liposomal/complexed antiinfectiveformulations were compared. In addition, a 10 fold higher dose of freeamikacin and a 10 fold higher dose of free tobramycin were also given.Dosing was IT daily over seven days. Results (FIG. 3) indicate thatliposomal/complexed amikacin in the two formulations (differing in lipidcomposition) revealed a significant reduction in CFU levels and werebetter at reducing CFUs than free amikacin or free tobramycin at 10-foldhigher dosages. In the Figure, Lip-An-14 is DPPC/Chol/DOPC/DOPG(42:45:4:9) and 10 mg/ml amikacin, Lip-An-15 is DDPC/Chol (1:1) also at10 mg/ml. All lipid-lipid and lipid-drug ratios herein are weight toweight.

The next experiment (FIG. 4) was designed to demonstrate the slowrelease and sustained antiinfective capabilities of liposomal/complexedamikacin. The dosing was every other day for 14 days, as opposed toevery day for seven days as in the previous experiments. Resultsindicate that liposomal/complexed amikacin in the two formulations(differing in lipid composition) had a 10 to 100 times more potent(greater ability to reduce CPU levels) than free amikacin or freetobramycin. A daily human dose of 600 mg TOBI® (or about 375 mg/m2)corresponds to a daily rat dose of 9.4 mg. Thus the data can be directlycorrelated to a 10 to 100 fold improvement in human efficacy. It shouldbe noted that a two-log reduction is the best that can be observed inthis model. A 100-fold reduction in P. aeruginosa in sputum assays hasbeen correlated with improved pulmonary function (Ramsey et al., 1993).The sustained release of the liposomal/complexed amikacin formulationsindicate that a lower dose and/or less frequent dosing can be employedto obtain a greater reduction in bacterial growth than can be obtainedwith free aminoglycoside.

The efficacy of liposomal/complexed amikacin was studied in a model forchronic pulmonary infection where P. aeruginosa was embedded in anagarose bead matrix that was instilled via the trachea of Sprague/Dawleyrats. Three days later free amikacin or liposomal/complexed amikacin wasdosed every day (FIG. 3) or every other day (FIG. 4) at 1 mg/rat or 10mg/rat of the given aminoglycoside or 1 mg/rat liposomal/complexedamikacin, as well as with blank liposomes (lipid vehicle) as thecontrol, with five rats per group.

The homogenized rat lungs (frozen) following the 14 day experiment wereanalyzed for aminoglycoside content and activity. The clinical chemicalassay was performed using a TDX instrument while the bioassay wasperformed by measuring inhibition zones on agar plates embedded withBacillus subtilis.

The results are shown in Table III:

Bioassay Clinical Assay Formulation (microgram/mL) (microgram/mL)Lip-An-14 at 10 mg/mL 9.5 9.1 Lip-An-15 at 10 mg/mL 21.5 18.4 Freeamikacin at 100 mg/mL nd 2.0 Free tobramycin at 100 mg/mL nd 1.4Drug weights are for the drug normalized to the absence of any saltform.

The Table III results indicate that aminoglycoside is present and activefor both liposomal/complexed antiinfective formulations, while littlecan be detected for the free aminoglycoside even at the 10-fold higherdose. These further results establish the sustained releasecharacteristics of liposomal/complexed antiinfective, and also confirmthat that antiinfective which remains is still active. Of the aboveformulations only the free tobramycin (0.1 microgram/ml) exhibited anydetectable levels of aminoglycoside in the kidneys.

The sustained release and depot effect of liposomal/complexed amikacinis further demonstrated in FIG. 5. Rats were given a chronic pulmonaryinfection where P. aeruginosa was embedded in an agarose bead matrixthat was instilled via the trachea, using the same beads employed in theefficacy studies. The rats were then given free tobramycin orliposomal/complexed amikacin (formulation Lip-An-14) via intratrachealadministration at the same dose (2 mg/rat). The data, measured inmicrogram antiinfective per gram lung tissue over time, show thatliposomal/complexed antiinfective exhibits a sustained release and depoteffect while free tobramycin revealed negligible levels in the lungs by24 hours, primarily due it is believed to rapid systemic absorption.This greater than a hundred-fold increase of antiinfective in the lungfor liposomal/complexed amikacin in an infected rat supports the idea ofa sustained release liposomal/complexed antiinfective that can be takensignificantly less often than the currently approved TOBI™ formulation.

The pharmacokinetics of amikacin was determined in rats followingintratracheal (IT) administration of either free tobramycin orliposomal/complexed amikacin. A dose of 2 mg/rat was administered. Thedepot effect of liposomal/complexed antiinfective technology isdemonstrated in that in comparison to free tobramycin given IT, agreater than a hundred-fold increase in drug for liposomal/complexedamikacin still remains in the infected lungs twenty-four hours followingadministration. Thus, the therapeutic level of drug is maintained for alonger period of time in the liposomal formulations compared to freetobramycin.

FIG. 7 shows remarkable residence time and accumulation of effectiveamounts of antiinfective in the lungs, a result that establishes thatrelatively infrequent dosings can be used. Each dose is 4 hr. byinhalation (in rat, 3 rats per group, as above) of nebulized liposomalamikacin (DPPC/Chol., 1:1) at 15 mg/ml amikacin. Dosing was at eitherday one; day one, three and five; or day one, two, three, four and five.Rats providing a given data bar were sacrificed after the respectivedosing of the data bar. The formulation is made as in the Example.

Similar anti-infectives can be utilized for the treatment ofintracellular infections like pulmonary anthrax and tularemia. Inpulmonary anthrax the anthrax spores reach the alveoli in an aerosol.The inhaled spores are ingested by pulmonary macrophages in the alveoliand carried to the regional tracheobronchial lymph nodes or mediastinallymph nodes via the lymphatics (Pile et al., 1998; Gleiser et al.,1968). The macrophage is central in the both the infective pathway andis the major contributor of host self-destruction in systemic(inhalation) anthrax. In addition to its attributes of sustained releaseand targeting, liposomal/complexed antiinfective technology can enhancecellular uptake and can use alveolar macrophages and lung epithelialcells in drug targeting and delivery. The possession of thesecharacteristics is believed to facilitate the treatment of theseintracellular infections, which infections occur in the lungs and aretransported by macrophages. More importantly; these characteristicsshould make the antiinfective more effective in that theliposomal/complexed antiinfective should be phagocytized by the verycells containing the disease. The antiinfective would be releasedintracellularly in a targeted manner, thereby attacking the infectionbefore it is disseminated. The encapsulated drug can be an alreadyapproved pharmaceutical like ciprofloxacin, tetracycline, erthyromycinor amikacin. Liposomal/complexed ciprofloxacin has been developed.

In a study this compound was administered to mice and compared to bothfree ciprofloxacin administered intratracheally and free ciprofloxacinadministered orally, with all three compounds given at the same dose(FIG. 6). The dose for each mouse was 15 mg/kg, with three mice pergroup. Liposomal/complexed cipro was in DPPC/Cholesterol (9:1), at 3mg/ml cipro, with the formulation produced as in the Example. The lipidto drug ratio was 12.5:1 by weight. In comparison to orally administeredciprofloxacin, liposomal/complexed ciprofloxacin was present in the micelungs at amounts over two orders of magnitude higher than freeciprofloxacin. Moreover, only liposomal/complexed ciprofloxacin showedlevels of drug in the lung after 24 hours, while the orally administereddrug was undetectable in less than two hours. This data supports the useof liposomal/complexed ciprofloxacin and other antiinfectives likeaminoglycosides, tetracyclines and macrolides for the treatment and forthe prophylactic prevention of intracellular diseases used bybioterrorists.

One type of process of manufacture of liposomal/complexed typicallycomprises ethanol infusion at room temperature, which is below the phasetransition temperature for the lipids used in the formulation. Liposomesin the form of small unilamellar vesicles (SUVs) are mixed with anaqueous or ethanolic solution containing the bioactive agent to beentrapped. Ethanol is infused into this mixture. The mixture immediatelyforms either extended sheets of lipid or multilamellar vesicles (MLVs).The extended sheets of lipid, if formed, can be induced form MLVs uponremoval of ethanol by either sparging or washing by such methods ascentrifugation, dialysis or diafiltration. The MLVs will typically rangein diameter between approximately 0.1 and approximately 3.0 μm.

Or, the lipids to be employed are dissolved in ethanol to form alipid-ethanol solution. The lipid-ethanol solution is infused in anaqueous or ethanolic solution containing the molecule of the bioactiveagent to be entrapped. All manipulations are performed below the phasetransition of the lowest melting lipid. The mixture immediately formseither extended sheets of lipid or multilamellar vesicles (MLVs)(10).The extended sheets of lipid will form MLVs upon removal of ethanol byeither sparging or washing by such methods as centrifugation, dialysisor diafiltration. The MLVs will typically range in diameter fromapproximately 0.1 to approximately 3.0 μm.

Lipids Mol ratio Lipid/amikacin, w/w DPPC — 1.1 DPPC/DOPG 9:1 1.0DPPC/DOPG 7:1 3.9 DPPC/DOPG 1:1 2.8 DPPC/DOPG 1:2 2.7 DOPG — 2.6DPPC/Cholesterol 19:1  1.0 DPPC/Cholesterol 9:1 1.2 DPPC/Cholesterol 4:11.7 DPPC/Cholesterol 13:7  2.1 DPPC/Cholesterol 1:1 2.7DPPC/DOPC/Cholesterol 8.55:1:.45 2.0 DPPC/DOPC/Cholesterol 6.65:1:.353.0 DPPC/DOPC/Cholesterol 19:20:1 2.5 DPPC/DOPG/Cholesterol 8.55:1:.453.8 DPPC/DOPG/Cholesterol 6.65:1:.35 4.1 DPPC/DOPG/Cholesterol 19:20:14.2 DPPC/DOPC/DOPG/Cholesterol 42:4:9:45 3.7 DPPC/DOPC/DOPG/Cholesterol59:5:6:30 3.7

A number of formulations with Amikacin were made by the method of theExample, as summarized below:

Further information of forming liposomal/complexed antiinfective can befound in PCT/US03/06847, filed Mar. 5, 2003, which is incorporatedherein by reference in its entirety.

Example

The following is a detailed description of the manufacture of 150 mL ofLiposomal/complexed amikacin.

Total Initial Volume=1.5 L

Ethanol Content=23.5% (v/v)Lipid Composition: DPPC/Chol (1:1 mole ratio)Intial [Lipid]=7.6 mg/mlIntial [amikacin sulfate]=57.3 mg/mlFinal product Volume=150 mL

I) Compounding and Infusion:

7.47 g DPPC and 3.93 g Cholesterol were dissolved directly in 352.5 mLethanol in a 50 C water bath. 85.95 g amikacin sulfate was dissolveddirectly in 1147.5 mL PBS buffer. The solution is then titrated with 10NNaOH or KOH to bring the pH to approximately 6.8.

352.5 mL ethanol/lipid was added or infused to the 1147.5 mLamikacin/buffer to give a total initial volume of 1.5 L. Theethanol/lipid was pumped @-30 mLmin (also called infusion rate) with aperistaltic pump into the amikacin/buffer solution which was beingrapidly stirred at 150 RPM in a reaction vessel on a stir plate at roomtemperature

The product was stirred at room temperature for 20-30 minutes.

II) Diafiltration or “Washing” Step:

The mixing vessel was hooked up to a peristaltic pump and diafiltrationcartridge. The diafiltration cartridge is a hollow membrane fiber with amolecular weight cut-off of 500 kilodaltons. The product was pumped fromthe reaction vessel through the diafiltration cartridge and then backinto the mixing vessel at room temperature. A back pressure ofapproximately 7 psi is created throughout the cartridge. Free amikacinand ethanol was forced through the hollow fiber membrane by the backpressure leaving the liposomal amikacin (product) behind. The productwas washed 8 times at room temperature. Fresh PBS buffer was added (viaanother peristaltic pump) to the reaction vessel to compensate for thepermeate removal and to keep a constant product volume.

The product was concentrated.

Publications and references, including but not limited to patents andpatent applications, cited in this specification are herein incorporatedby reference in their entirety in the entire portion cited as if eachindividual publication or reference were specifically and individuallyindicated to be incorporated by reference herein as being fully setforth. Any patent application to which this application claims priorityis also incorporated by reference herein in the manner described abovefor publications and references.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations in the preferred devices and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the claims that follow.

REFERENCES

-   1. Veldhuizen, R., Nag, K., Orgeig, S. and Possmayer, F., The Role    of Lipids in Pulmonary Surfactant, Biochim. Biophys. Acta    1408:90-108 (1998).-   2. Hagwood, S., Derrick, M. and Poulain, F., Structure and    Properties of Surfactant Protein B, Biochim. Biophys. Acta    1408:150-160 (1998).-   3. Johansson, J., Structure and Properties of Surfactant ProteinC,    Biochim. Biophys. Acta 1408:161-172 (1998).-   4. Ikegami, M. and Jobe, A. H., Surfactant Protein Metabolism in    vivo, Biochim. Biophys. Acta 1408218-225 (1998).-   5. Couveur, P., Pattel, E. and Andremont, A., Liposomes and    Nanoparticles in the Treatment of Intracellular Bacterial    Infections, Pharm. Res. 811079-1085 (1991).-   6. Gonzales-Rothi, R. J., Casace, J., Straub, L., and Schreier, H.,    Liposomes and Pulmonary Alveolar Macrophages: Functional and    Morphologic Interactions, Exp. Lung Res. 17:685-705 (1991).-   7. Swenson, C. E., Pilkiewicz, F. G., and Cynamon, M. H., Liposomal    Aminoglycosides and TLC-65 Aids Patient Care 290-296 (December,    1991).-   8. Costerton, J. W., Stewart, P. S., and Greenberg, E. P., Bacterial    Biofilms: A Common Cause of Persistent Infections, Science    2841318-1322 (1999).-   9. Cash, H. A., Woods, D. E., McCullough, W. G., Johanson, J. R.,    and Bass, J. A., A Rat Model of Chronic Respiratory Infection with    Pseudomonas aeruginosa, American Review of Respiratory Disease    119:453-459 (1979).-   10. Cantin, A. M. and Woods, D. E. Aerosolized Prolastin Suppresses    Bacterial Proliferation in a Model of Chronic Pseudomonas aeruginosa    Lung Infection, Am. J. Respir. Crit. Care Med. 160:1130-1135 (1999).-   11. Ramsey, B. W., Dorkin, H. L., Eisenberg, J. D., Gibson, R. L.,    Harwood, I. R., Kravitz, R. M., Efficacy of Aerosolized Tobramycin    in Patients with cystic Fibrosis. New England J. of Med.    328:1740-1746 (1993).-   12. Mendelman, P. M., Smith, A. L, Levy, J., Weber, A., Ramsey, B.,    Davis, R. L., Aminoglycoside Penetration, Inactivation, and Efficacy    in Cystic Fibrosis Sputum, American Review of Respiratory Disease    132:761-765 (1985).-   13. Price, K. E., DeFuria, M. D., Pursiano, T. A. Amikacin, an    aminoglycoside with marked activity against antibiotic-resistant    clinical isolates. J Infect Dis 134:S249-261 (1976).-   14. Damaso, D., Moreno-Lopez, M., Martinez-Beltran, J.,    Garcia-Iglesias, M. C. Susceptibility of current clinical isolates    of Pseudomonas aeruginosa and enteric gram-negative bacilli to    Amikacin and other aminoglycoside antibiotics. J Infect Dis    134:S394-90 (1976).-   15. Pile, J. C., Malone, J. D., Eitzen, E. M., Friedlander, A. M.,    Anthrax as a potential biological warfare agent. Arch. Intern. Med.    158:429-434 (1998).-   16. Gleiser, C. A., Berdjis, C. C., Hartman, H. A., &    Glouchenour, W. S., Pathology of experimental respiratory anthrax in    Macaca mulatta. Brit. J. Exp. Path., 44:416-426 (1968).

1.-25. (canceled)
 26. A liposomal aminoglycoside formulation comprisingan aminoglycoside or salt thereof encapsulated in liposomes, wherein thelipid component of the liposomes consists of a sterol and aphosphatidylcholine, and the weight ratio of the lipid component toaminoglycoside is less than 2.5 (lipid component) to 1 (aminoglycoside).27. The liposomal aminoglycoside formulation of claim 26, wherein theaminoglycoside is streptomycin, gentamicin, tobramycin, amikacin,netilmicin or kanamycin, or a salt thereof.
 28. The liposomalaminoglycoside formulation of claim 26, wherein the aminoglycoside isamikacin, or a salt thereof.
 29. The liposomal aminoglycosideformulation of claim 28, wherein the aminoglycoside or salt thereof isamikacin sulfate.
 30. The liposomal aminoglycoside formulation of claim26, wherein the phosphatidylcholine is selected from the groupconsisting of egg phosphatidylcholine (EPC), soy phosphatidylcholine(SPC), hydrogenated egg phosphatidylcholine (HEPC), hydrogenated soyphosphatidylcholine (HSPC), dipalmitoyl phosphatidylcholine (DPPC),dioleoyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine(DMPC), distearoyl phosphatidylcholine (DSPC), palmitoylstearoylphosphatidylcholine (PSPC), and mixtures thereof.
 31. The liposomalaminoglycoside formulation of claim 26, wherein the phosphatidylcholineis DPPC.
 32. The liposomal aminoglycoside formulation of claim 26,wherein the sterol is cholesterol.
 33. The liposomal aminoglycosideformulation of claim 26, wherein the sterol is cholesterol and thephosphatidylcholine is DPPC.
 34. The liposomal aminoglycosideformulation of claim 28, wherein the sterol is cholesterol and thephosphatidylcholine is DPPC.
 35. The liposomal aminoglycosideformulation of claim 29, wherein the sterol is cholesterol and thephosphatidylcholine is DPPC.
 36. A method of treating a mycobacterialinfection in the lungs of a patient in need thereof, comprisingadministering to the lungs of the patient an effective amount of theformulation of claim
 1. 37. The method of claim 36, wherein theaminoglycoside is streptomycin, gentamicin, tobramycin, amikacin,netilmicin or kanamycin, or a salt thereof.
 38. The method of claim 36,wherein the aminoglycoside is amikacin, or a salt thereof.
 39. Themethod of claim 38, wherein the aminoglycoside or salt thereof isamikacin sulfate.
 40. The method of claim 36, wherein thephosphatidylcholine is selected from the group consisting of eggphosphatidylcholine (EPC), soy phosphatidylcholine (SPC), hydrogenatedegg phosphatidylcholine (HEPC), hydrogenated soy phosphatidylcholine(HSPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC),distearoyl phosphatidylcholine (DSPC), palmitoylstearoylphosphatidylcholine (PSPC), and mixtures thereof.
 41. The method ofclaim 36, wherein the phosphatidylcholine is DPPC.
 42. The method ofclaim 36, wherein the sterol is cholesterol.
 43. The method of claim 38,wherein the sterol is cholesterol and the phosphatidylcholine is DPPC.44. The method of claim 39, wherein the sterol is cholesterol and thephosphatidylcholine is DPPC.
 45. The method of claim 36, whereinadministering to the lungs of the patient comprises inhalationadministration via a nebulizer at a dosing schedule of once a day orless.
 46. The method of claim 36, wherein the mycobacterial infection isa M. avium complex (M. avium and M. intracellulare) infection.